NMR STRUCTURE OF THE NANOS METAL BINDING DOMAIN

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Nanos [nos], is a posterior group gene essential for abdominal development in Drosophila. Nanos protein exerts its morphogenic effects by inhibiting protein expression from maternal transcripts of the hunchback [hb] gene. Two nearly identical 11 nucleotide repeat sequences in the 3' untranslated region (3' UTR) of hb mRNA are required for the nanos effect, and these repeated sequences are referred to as nanos response elements (NREs). The nanos C-terminal domain (approximately 120 residues) is highly conserved among Dipteran insects. It has been proposed that the nanos C-terminal domain contains two CCHC zinc binding motifs which recognize NREs. We will characterize the specificity, stoichiometry, affinity and cooperativity of metal binding to the nanos C-terminal domain. By preparing various truncated versions of the C- terminal domain, the minimal metal- and NRE-binding sequence of nanos will be determined. The tertiary structure of the minimal C-terminal domain that has optimal metal and RNA binding activities will be studied using multi-dimensional nuclear magnetic resonance (NMR). Based upon the structure of nanos C-terminal domain, hypotheses will be made for mutational studies using in vivo and in vitro assays by collaboration with Prof. Ruth Lehmann at the Whitehead Institute. These studies will establish the foundation for the long term goal -- solving the nanos/NRE complex structure to elucidate the structural basis for their specific interactions.